In recent years, a number of proteins have come into use as pharmaceuticals. This is because the gene recombination technology has been developed for and applied to the introduction or transfer of a gene coding for a desired protein into microorganisms or mammalian cells, so that commercial protein production is now feasible by cultivating the thus-produced genetically modified organisms. For such a medicinal protein to show the physiological activity or activities intrinsic therein, posttranscriptional modifications, for example folding, glycosylation and disulfide bond formation is necessary as in nature.
Methods of producing proteins by cultivating microorganisms are capable of producing proteins at low costs since microorganisms can grow rapidly and medium compositions therefor are simple. However, in many cases, due posttranscriptional modifications of the desired protein are not made properly in microorganisms. Therefore, it is difficult to obtain a protein having the same physiological activity as that of the natural counterpart in sufficient quantities; in the existing circumstances, it is still a long way to practical use of such protein production methods on a commercial basis.
Therefore, it is the mainstream of the art to introduce a gene for a desired protein into mammalian cells and cultivating the cells to cause them to product the protein. Such pharmaceutical proteins as blood coagulation factors, thrombolytic agents and antibodies for pharmaceutical use as produced using recombinant mammalian cells are already on the market and used. However, those methods which use mammalian cells have a problem in that culture tanks and medium for exclusive use are required and the production cost is high.
To overcome these problems, animal factories have now attracted attention. The technology concerned comprises using gene-transferred (transgenic) animals to produce desired proteins. Attempts have been made to produce transgenic mammals using goats, sheep and cows, among others, and cause the production of the desired proteins in the milks thereof. Thus, there is a report describing the expression of an antibody at a level of 10 mg/ml in milk, although the expression level varies depending on the protein species (cf. e.g. Non-Patent Document 1). However, this technology has the BSE (bovine spongiform encephalopathy) problem and other problems; utilizable mammalian individuals are large-sized and, therefore, are difficult to produce, raise and handle; a further problem is that the period from birth to sexual maturation is long, namely 8 months in goats or sheep, or 15 months in cows.
Therefore, investigations have been made to use transgenic birds for the expression of a desired protein in eggs thereof. This technology has several advantages: the egg-laying productivity is high, there is no BSE problem, the maturation period is short (5 months in chickens), individuals are small in size and therefore a large number of individuals can be raised, the technique of artificial insemination has been established, enabling rapid raising of large-scale transgenic groups, and the egg inside is generally sterile by nature.
As for the methods of producing transgenic birds, the method using a retrovirus vector, the method using embryonic stem cells, the method using primordial germ cells and the method comprising causing a target gene to adhere to spermatozoa for introduction thereof, among others, are under investigation. Among those methods, the method using a retrovirus vector is the commonest. So far, a study in which an avian leukemia virus (ALV)-derived replication defective retrovirus vector was used has been reported (cf. e.g. Patent Document 1). The target protein used was β-lactamase, and the promoter gene used was the cytomegalovirus (CMV) promoter gene. Transgenic birds were produced successfully by retrovirus vector introduction into blastoderms at the stage X just after egg laying. Reportedly, the level of expression was 0.33 mg/ml (the egg white volume being estimated at 40 ml) as determined by western blot analysis and, when expressed in terms of β-lactamase activity, it was 0.003 to 0.033 mg/ml. On that occasion, the frequency of appearance of G0 transgenic chimeric birds was 20%. The result of an investigation of the efficiency of introduction into germ cells indicated that about 5% of male G0 transgenic chimeric birds had the transgene in spermatozoa. According to another report about a similar experiment, the transgene expression was about 1.2 μg/ml of egg white in G2 birds having the transgene introduced in the whole body (cf. e.g. Non-Patent Document 2). On that occasion, the frequency of appearance of G1 from G0 was 3/422 (0.71%).
Further, there are reports about transgenic birds expressing human interferon or human-derived erythropoietin (cf. e.g. Patent Document 2 and 3). Interferon is a glycoprotein having a molecular weight of about 20,000 which is produced and secreted by almost all animal cells on the occasion of viral infection; it is also known as virus inhibiting factor. Erythropoietin (EPO) is a sugar chain-rich polypeptide mainly produced in the kidney and capable of acting on precursor cells in the hemopoietic tissue to promote the differentiation thereof into and the growth of erythrocytes. Currently, recombinant human EPO produced by the recombinant DNA technology using animal cells as hosts is on the market and is used mainly as a therapeutic agent for various types of anemia, typically renal anemia resulting from nephropathy-associated reduced EPO productivity. When an ALV-derived replication defective retrovirus vector and the CMV promoter gene or ovomucoid-ovotransferrin fused promoter gene were used, human interferon was expressed in serum at a maximum level of 200 ng/ml, and human-derived erythropoietin in serum and egg white each at a maximum level of 70 ng/ml.
In another report, it is reported that high levels of virus titer, infectivity and expression were obtained using the mouse stem cell virus (MSCV) vector and VSV-G envelope (cf. e.g. Patent Document 4). Further, according to that report, high expression levels were realized by adjusting the time of retrovirus vector introduction and, when an anti-prion single chain antibody (scFv) is used as the target protein, high levels of expression of 0.5 to 1 mg/ml in egg white and in egg yolk were realized.
Cats are animals long loved as pets by humans and recently have been establishing their position as the so-called “partner, companion or friend animals” in the human society. On the other hand, in the fields of medicine, pharmacology, veterinary medicine and psychology, among others, cats have so far been used as experimental animals and recently have come into use in testing pharmaceuticals for safety and efficacy. In view of the circumstances in which the social importance of cats is increasing, feline diseases and infections are objects of concern and effective therapeutic means therefor are desired. In recent years, medicinal proteins have attracted attention in the treatment of feline diseases as well and, currently, medicinal proteins for human use are mainly used in cats as well. However, medicinal proteins for human use differ in amino acid sequence from in vivo proteins intrinsic in cats and, therefore, may possibly differ in effect or efficacy in living cats. Further, the difference in amino acid sequence may possibly cause an allergic reaction and, in the worst case, an anaphylactic symptom. Thus, such proteins cannot be used in high-frequency dosage regimens, so that the development of medicinal proteins intrinsic in cats is demanded.
As the feline-derived medicinal proteins so far studied widely, there may be mentioned cytokines. Cytokines are proteinic factors which are released from cells and mediate intercellular interactions in the exertion of immune or inflammatory response modulating, antiviral, antitumor, and cell proliferation and differentiation regulating actions. As the feline-derived cytokines so far reported, there may be mentioned erythropoietin (cf. e.g. Non-Patent Document 3 and 4) and interleukin 12 (cf. e.g. Patent Document 5), among others. As regards the production of these, mammalian cells have so far been used; under the existing circumstances, any transgenic birds have been used in such production.
Patent Document 1: Japanese Kohyo Publication 2001-520009
Patent Document 2: United States Patent Application Publication 2004/0019922
Patent Document 3: United States Patent Application Publication 2004/0019923
Patent Document 4: Japanese Kokai Publication 2002-176880
Patent Document 5: International Publication WO 97/046583
Non-Patent Document 1: Trends Biotechnol. 1999, Sep.; 17(9):367-74
Non-Patent Document 2: Nat. Biotechnol. 2002, Apr.; 20(4):396-9
Non-Patent Document 3: Blood, 1993, Sep. 1; 82(5):1507-16
Non-Patent Document 4: Vet. Immunol. Immunopathol. 1986, Jan.; 11(1):1-19